BM Levels Research Articles

The parasitic worm product ES-62 protects against collagen-induced arthritis by resetting the gut-bone marrow axis in a microbiome-dependent manner.

The parasitic worm-derived immunomodulator, ES-62 rescues defective levels of IL-10-producing regulatory B cells (Bregs) and suppresses chronic Th1/Th17-driven inflammation to protect against joint destruction in the mouse collagen-induced arthritis (CIA) model of rheumatoid arthritis. Such autoimmune arthritis is also associated with dysbiosis of the gut microbiota and disruption of intestinal barrier integrity. We recently further exploited the CIA model to show that ES-62's prevention of joint destruction is associated with protection of intestinal barrier integrity and normalization of the gut microbiota, thereby suppressing the gut pathology that precedes the onset of autoimmunity and joint damage in CIA-mice. As the status of the gut microbiota impacts on immune responses by influencing haematopoiesis, we have therefore investigated whether ES-62 harnesses the homeostatic mechanisms regulating this gut-bone marrow (BM) axis to resolve the chronic inflammation promoting autoimmunity and joint destruction in CIA. Reflecting this, ES-62 was found to counteract the BM myeloid/lymphoid bias typically associated with chronic inflammation and infection. This was achieved primarily by ES-62 acting to maintain the levels of lymphoid lineages (B220+ and CD3+ cells) observed in naïve, healthy mice but lost from the BM of CIA-mice. Moreover, ES-62's ability to prevent bone-destroying osteoclastogenesis was found to be associated with its suppression of CIA-induced upregulation of osteoclast progenitors (OCPs) in the BM. Critically, and supporting ES-62's targeting of the gut-BM axis, this rewiring of inflammatory haematopoiesis was lost in mice with a depleted microbiome. Underlining the importance of ES-62's actions in restoring steady-state haematopoiesis, the BM levels of B and T lymphoid cells were shown to be inversely correlated, whilst the levels of OCPs positively correlated, with the severity of joint damage in CIA-mice.

Relation of junctional adhesion molecule-a in multiple myeloma patients with possible prognostic factors

Abstract Background Multiple myeloma (MM) is a clonal plasma cell disorder characterized by the abnormal increase of monoclonal paraprotein leading to specific end-organ damage. Drug resistance is one of the major challenges in treating MM. As an emerging cell adhesion-mediated drug resistance (CAM-DR) agent, JAM-A (Junctional adhesion molecule-A), represents an exciting new potential drug target and biomarker for treating MM. However, the pathological role and clinical relevance of JAM-A in MM remains ambiguous. Aim To study the relation of JAM-A expression levels in MM patients with possible prognostic factors. Patients and methods The study was carried out on a total of 100 subjects divided into two groups. The first group included 50 multiple myeloma patients, while the second group included 50 patients performing aspiration of bone marrow for other conditions with normal plasma cell percentage. All subjects in the current study were subjected to full history taking and complete clinical examination, radiological studies, routine laboratory investigations, bone marrow aspiration/ biopsy, immunophenotyping, measurement of serum IL-6 levels and estimation of JAM-A expressions by Quantitative real-time PCR. Results JAM-A was overexpressed in newly diagnosed MM patients compared with post-treated patients and control group. Moreover, high JAM-A expression levels showed statistically significant correlations with plasma cell percentage in BM, serum β2-microglobulin and serum IL-6 levels. Conclusion JAM-A is overexpressed in MM and could be used as innovative prognostic biomarker.

Ovariectomy induces bone loss via microbial-dependent trafficking of intestinal TNF+ T cells and Th17 cells.

Estrogen deficiency causes a gut microbiome-dependent expansion of BM Th17 cells and TNF-α-producing T cells. The resulting increased BM levels of IL-17a (IL-17) and TNF stimulate RANKL expression and activity, causing bone loss. However, the origin of BM Th17 cells and TNF+ T cells is unknown. Here, we show that ovariectomy (ovx) expanded intestinal Th17 cells and TNF+ T cells, increased their S1P receptor 1-mediated (S1PR1-mediated) egress from the intestine, and enhanced their subsequent influx into the BM through CXCR3- and CCL20-mediated mechanisms. Demonstrating the functional relevance of T cell trafficking, blockade of Th17 cell and TNF+ T cell egress from the gut or their influx into the BM prevented ovx-induced bone loss. Therefore, intestinal T cells are a proximal target of sex steroid deficiency relevant for bone loss. Blockade of intestinal T cell migration may represent a therapeutic strategy for the treatment of postmenopausal bone loss.

Prognostic significance of Wilms tumor 1 mRNA expression levels in peripheral blood and bone marrow in patients with myelodysplastic syndromes.

This present study was designed to follow up 82 patients among 115 MDS patients registered in study ODK-0801 for 5 years, to analyze the relationship between the WT1 mRNA expression level and prognosis. This study aimed to investigate the clinical utility of WT1 mRNA expression levels. After study ODK-0801, we investigated the conditions of the same patients once a year, including any clinical and laboratory findings supporting the diagnosis, and treatment among the living patients. When we assessed the survival time of 82 MDS patients by WT1 mRNA expression level, there were significant differences between the < 500 and ≥ 104 copies/μ g RNA groups and between the 500-104 and ≥ 104 copies/μ g RNA groups for BM levels (p < 0.01). Examination of the time of freedom from acute myeloid eukemia (AML) transformation indicated that a high WT1 mRNA expression level (> 104 copies/μ g RNA) was a strong prognostic factor for a short time to AML transformation. The results indicate that the tumorigenesis of MDS is likely to originate at the stem cell level, suggesting that the WT1 mRNA level measurement in the BM is an effective prognostic marker in patients with MDS.

Hematopoietic, Mesenchymal, and Immune Cells Are More Enhanced in Bone Marrow than in Peripheral Blood from Granulocyte Colony-Stimulating Factor Primed Healthy Donors

The use of granulocyte colony-stimulating factor (G-CSF) primed bone marrow (G-BM) has been recently considered as an alternative to mobilized hematopoietic stem cells from peripheral blood (G-PB), especially in the haploidentical transplant setting. The purpose of this study was to compare the effect of in vivo G-CSF priming on BM and PB hematopoietic, mesenchymal (MSC), and immune cells. Forty healthy donors undergoing BM harvest for haploidentical transplant were given subcutaneous recombinant human G-CSF for 7 days. BM and PB samples were harvested on days −7 and 0. The hematopoietic stem/progenitor cells increased significantly after G-CSF priming in both BM and PB with a selective rise of BM CD34+CD38− cell subset. A striking enhancement of the mesenchymal progenitors was detected in G-BM. CD3+, CD4+, CD8+, and CD19+ cell fractions; the naive CD4+ and CD8+ subpopulations; and natural killer and regulatory T cells increased in G-BM, whereas only slight changes were detected in PB. Myeloid dendritic cells (DC1) were significantly up-regulated in both G-BM and G-PB, whereas DC2 increased only in G-BM. In conclusion, our results show substantial differences in the biologic effects exerted by G-CSF at BM and PB levels on hematopoietic cells and immune cell fractions. Furthermore, the impressive rise of MSC progenitors in G-BM might also be relevant to provide MSCs for several clinical use.

Prognostic Impact of Circulating Plasma Cells on Survival of Patients With Multiple Myeloma

482 patients with monoclonal gammopathy including MGUS, smoldering MM and symptomatic MM patients. X-ray survey, positron emission tomography (PET)/computerized tomography (CT) and magnetic resonance imaging (MRI) were performed in MM patients to define the presence of bone disease. Any significant correlation between osteolytic lesions and cytogenetic profiles was not found with the exception for the presence of t(11;14). On the other hand significant higher BM median levels of both CCL3 and CCL20 were found in MM patients with osteolytic lesions at X-rays as compared to those negative and in PET/CT positive patients as compared to those negative. Moreover the BM levels of CCL3, CCL20, Activin-A and DKK-1 were significantly higher in high bone disease patients as compared to low bone disease ones whereas significant higher BM median levels of Activin A and CCL20 were found in MM patients with bone fractures as compared to those without fractures. By a logistic multivariate analysis, we found that BM CCL20 levels were significant predictors of osteolysis at the Xrays. In relationship with the presence of lesions at the MRI in all MM patients we found that BM median levels of CCL3, CCL20, Activin-A and DKK-1 were significantly higher although only those of DKK-1 were related to the presence of MRI lesions independently to the osteolysis at the X-rays. Our data define the relationship between bone disease and the molecular and BM cytokine and chemokine features suggesting the role of BM CCL20 levels as a new marker of osteolysis in MM patients.

An Adhesive Bone Marrow Scaffold and Bone Morphogenetic-2 Protein Carrier for Cartilage Tissue Engineering

A chondroitin sulfate-bone marrow (CS-BM) adhesive hydrogel was used to localize rhBMP-2 to enhance articular cartilage tissue formation. Chondrocyte pellet culture revealed that 0.1 and 1 μg/mL of rhBMP-2 enhanced sulfated-GAG content. rhBMP-2 localization within the hydrogels was investigated, and it was found that BM, CS-NHS, and rhBMP-2 levels and time affected rhBMP-2 retention. Retention was modulated from 82 to 99% over a 3-week period for the material formulations investigated. To evaluate carrier efficacy, rhBMP-2 and bovine articular chondrocytes were encapsulated within CS-BM, and biochemical evaluation revealed significant increases in total collagen production with rhBMP-2. Histological analysis revealed more robust tissue formation and greater type-II collagen production with encapsulated rhBMP-2. Subsequently, a subcutaneous culture of hydrogels revealed increased total collagen, type-II to type-I collagen ratio, and sulfated GAG in samples carrying rhBMP-2. These findings indicate the development of a multifunctional system capable of localizing rhBMP-2 to enhance repair tissue quality.

Enhancement of Megakaryocyte Interactions with the Osteoblastic Hematopoietic Stem Cell Niche Improves Engraftment Efficiency Following Hematopoietic Stem Cell Transplantation

Abstract 211Bone marrow (BM) radioablation produces structural changes in the endosteal osteoblastic stem cell niche, a critical site of hematopoietic stem cell (HSC) engraftment following HSC transplantation (HSCT). We have previously shown that total body irradiation (TBI) in wildtype (WT) mice induces migration of recipient megakaryocytes to the niche and an expansion of niche osteoblasts that supports HSC engraftment following transplantation. We have also demonstrated that c-MPL-deficient (mpl−/−) recipients have decreases in total megakaryocytes (35% of WT), the percentage of megakaryocytes migrating to the endosteum (<20% of WT), and niche osteoblast expansion (<50% of WT) following TBI, leading to profound deficits in long-term (LT)-HSC engraftment following HSCT. We now present data examining mechanisms by which megakaryocytes facilitate both niche osteoblast expansion post-TBI and donor HSC engraftment following HSCT, and a therapeutic strategy utilizing these mechanisms to enhance donor HSC engraftment.The decrease in total megakaryocytes and absent thrombopoietin (TPO) signaling in mpl−/− mice resulted in a 90% reduction in post-TBI mpl−/− versus WT BM levels of platelet-derived growth factor beta (PDGFβ), a known osteoblast growth factor. In vitro, megakaryocytes cultured together or across a transwell membrane markedly enhanced osteoblast growth (> 2.5 fold, p < 0.001), but PDGFβ signaling inhibition completely abrogated megakaryocyte-driven osteoblast growth. In vivo, inhibition of PDGF receptor signaling in WT mice via imatinib treatment resulted in near complete blockade of TBI-induced osteoblast expansion, and imatinib treatment of primary recipients resulted in diminished LT-HSC engraftment in secondary transplant assays.Blockade of CD41 integrin-mediated adhesion of megakaryocytes in WT recipient BM blocked TBI-induced megakaryocyte migration to the endosteal niche and severely abrogated LT-HSC engraftment efficiency. However, in contrast to c-MPL deficiency, CD41 blockade did not decrease PDGFβ expression or niche osteoblast expansion, suggesting that in addition to PDGFβ-dependent effects on niche expansion, the megakaryocyte migration to the niche itself is also required to efficiently engraft HSC. Mice with decreased GATA-1 expression (Gata-1tm2sho/J), have a large increase in total BM megakaryocytes a >2-fold (p < 0.001) increase in PDGFβ levels, and greatly increased expansion of osteoblast and other mesenchymal elements 48 hours post-TBI compared to WT mice. However, Gata-1tm2sho/J megakaryocytes have known defective terminal differentiation and function including decreased platelet production, and Gata-1tm2sho/J primary recipients did not engraft LT-HSC more efficiently than WT primary recipients, demonstrating the need for fully functional megakaryocytes, and not only increased PDGFβ-induced mesenchymal proliferation, to foster HSC engraftment.Finally, we have examined whether TPO administration prior to radioablation and HSCT can enhance host megakaryocyte effects on the niche and HSC engraftment. TPO administration for 5 days prior to radioablation, resulted in a significant increase in BM megakaryocytes and a 50% increase in niche osteoblast expansion. Furthermore, competitive secondary transplantation assays demonstrated that TPO- versus sham-treatment of primary recipients prior to TBI and BM transplant, resulted in increased initial engraftment at 24 hours post-primary transplant (40% increase, p < 0.05) increased short-term HSC and progenitor engraftment 3–6 weeks following secondary transplant (4–20 fold increase, p < 0.02), and sustained LT-HSC engraftment at 28 weeks post-transplant in 47% versus 7% (p < 0.05) of secondary recipients of TPO- versus sham-treated primary recipient BM, respectively.Taken together, our results demonstrate that host megakaryocytes facilitate efficient HSC engraftment following TBI and HSCT through PDGFβ-dependent enhancement of niche osteoblast expansion and through direct interactions of megakaryocytes with the niche. TPO-treatment of transplant recipients prior to radioablation and stem cell infusion enhances these megakaryocyte-dependent pathways and subsequent donor HSC engraftment efficiency, providing a clinically applicable strategy to enhance niche function and stem cell engraftment following clinical transplantation. Disclosures:No relevant conflicts of interest to declare.

Effects of everolimus (EVE) on disease progression in bone and bone markers (BM) in patients (pts) with bone metastases (mets).

512 Background: BOLERO-2, a multinational, double-blind, placebo-controlled, phase III study in postmenopausal women with estrogen-receptor–positive breast cancer (BC) refractory to non-steroidal aromatase inhibitors (NSAIs), showed significant clinical benefits with the addition of EVE to exemestane (EXE) (Baselga J, et al. NEJM. 2011 Epub). As bone resorption is an important factor in BC mets, it is interesting to study bone-related effects of EVE. In preclinical studies, mTOR inhibition was associated with decreased osteoclast survival and activity. Exploratory analyses in BOLERO-2 evaluated the effect of EVE vs placebo (PBO) on BM levels and BC progression in bone in pts with bone mets at baseline. Methods: Eligible pts were treated with EXE (25 mg once daily) and randomized (2:1) to EVE (10 mg once daily) or PBO. Bone turnover markers were exploratory endpoints analyzed at 6 and 12 wks after treatment initiation, and included bone-specific alkaline phosphatase, amino-terminal propeptide of type I collagen, and C-terminal cross-linking telopeptide of type I collagen. Progressive disease in bone (PDB) was defined as worsening of a pre-existing bone lesion or a new bone lesion. Results: Baseline disease characteristics, including bone mets at baseline (n = 370, 76% EVE vs n = 184, 77% PBO), were well balanced between arms (N = 724), and baseline bisphosphonate use was not (44% EVE vs 55% PBO). At 12.5 mo median follow-up, progression-free survival (primary endpoint), overall response rate, and clinical benefit rate (P &lt; .0001, all) were significantly higher with EVE (n = 485) vs PBO (n = 239). BM levels at 6 and 12 wks increased vs baseline with PBO, but decreased with EVE. The cumulative incidence rate of BC PBD was lower for EVE vs PBO at day 60 (3.03% vs 6.16%, respectively) and this trend was sustained beyond 6 months. Updated results will be presented. All bone-related adverse events reported were grade1/2 and occurred with similar frequency in EVE- (2.9%) and PBO- (3.8%) treated patients. Conclusions: Exploratory analyses from BOLERO-2 suggest that adding EVE has beneficial effects on bone turnover and BC progression in bone in pts receiving EXE therapy for NSAI-refractory BC.

Behavioural and physiological correlates of personality in greylag geese (Anser anser)

Personality means suites of correlated behavioural traits, also referred to as “behavioural syndromes” or “personality dimensions”. Across animal taxa similar combinations of traits seem to prevail, which may have proximate foundation in common neuroendocrine mechanisms. Hitherto, these have been rarely studied in intact social settings. We investigated personalities of greylag goose males from a free-roaming flock that shows complex social relationships. In connection with our longitudinal study on the consistency of behavioural and physiological responses to multiple challenges, we asked whether and how single, personality-related behavioural traits correlate with each other to form personality dimension(s). We tested whether these dimensions were related to physiological characteristics that previously showed limited plasticity (heart rate (HR), baseline and stress-induced excreted immuno-reactive corticosterone (BM), and testosterone metabolites levels) and, furthermore, to age, body measures, and dominance rank. Principal-components analysis based on behavioural variables revealed two factors: 51.1% of variability was explained by “aggressiveness” and a further 19.1% by “sociability”. “Aggressiveness” comprised correlated measures of aggression, subordinance, boldness, vigilance, and proximity to the mate. This “aggressiveness” positively correlated with stress-induced BM levels, the HR increase during aggressive interactions, and with dominance rank, which may suggest proximate and functional contingencies of this personality dimension.

A187 T-Cell Depleted RIC Allogeneic Stem Cells Transplantation: A Platform for Immunotherapy

who underwent bortezomib treatment. Methods: We studied BM levels of thrombospondin in 55 MM patients (30 male/25 female, median age: 65.1 years; median follow-up time, 18.8 months) treated with bortezomib. BM levels were measured in 25% (14/55) MM patients in time of diagnosis, in 35% (19/55) MM patients in first relapse and in 40% (22/55) MM patients in second and next relapse using ELISA. Eligible patients were divided into 3 groups according to treatment response: group (A) 42% (21/50) patients who achieved at least very good partial response (VGPR), including 24% (12/50) patients who achieved complete remission (CR); group (B) 26%(13/50) patients who achieved partial response (PR); group (C) 34% (17/50) patients who did not achieve even PR. Correlations between levels of thrombospondin and treatment response were assessed using the t test and Anova. Results: Statistically significant differences were found between group A (n = 21; median = 582.0 pg/L; 232-816) group B (n = 13; median = 400 pg/L; 103-672) and group C (n = 17; median = 285.0 pg/L; 96-572) P < .001, if compared together with Anova. Also, thrombospondin concentration is significantly higher at group A than in others (n = 30; median = 337 pg/L; 103-672) P < .001. Similar thrombospondin is significantly higher in patients with CR (n = 12; median = 573.0 pg/L; 406-816.) than in the others (n = 39; median = 400 pg/L; 103-612) P = .002. Conclusion: Findings indicate that low pretreatment thrombospondin levels in MM patients treated with bortezomib are associated with unfavorable response. It means, that angiogenesis is more inhibited in patients with CR and VGPR than in others. Treatment response is strong prognostic factor and BM thrombospondin levels at diagnosis time correlate closely with it. Such as they could became new prognostic and predictive factors. Supported by MSMT0021622434,LC06027.

Predictive Value of Minimal Residual Disease (MRD) Monitoring by RQ-PCR in WT1 Positive Patients Entered in the UK MRC AML-15 Trial

Relapse remains the main cause of treatment failure in Acute Myeloid Leukaemia (AML). Some 65% of AML patients lack a specific fusion transcript which can serve as a molecular target for detecting residual disease. However, in over 75% of AML cases, the Wilm's Tumor (WT1) gene is over-expressed and can therefore be used for MRD monitoring by RQ-PCR. The clinical value of serial MRD monitoring in WT1 positive patients was prospectively assessed in the AML-15 Trial which opened in 2002. The trial compared 3 induction regimens (DA v/s ADE v/s FLAG Ida), followed by randomisation in consolidation (courses 3 and 4) to either MACE + MidAC or 2 doses of Ara-C (3g/m2 or 1.5g/m2) and to stop or have a 5th course (Ara-C 1.5g/m2). Patients were also randomised to receive Gemtuzumab Ozogamicin (3mg/m2) at induction and/or consolidation. Adults in the standard and poor risk groups were scheduled to receive an allograft if a matched donor was available. An “in house” WT1 assay used primers/probes covering exons 7 and 8 in the 3′ region of the WT1 gene. WT1 transcripts from BM and PB were measured by real-time quantitative PCR (RQ-PCR) on the 7900 HT ABI machine, at diagnosis, after each course of chemotherapy, stem cell transplant, and during remission for up to 3 years. WT1 copies were normalised to ABL gene and expressed per 105ABL copies. WT1 was over-expressed in >80% of AML cases screened, when compared to normal BM copies (median 544, range 31–1173), PB (median 10, r 0–58), PBSC (median 71, r 0–483). In this study, 700 patients (excluding Core Binding Factor positive AML) with diagnostic BM WT1 levels >104 copies have been recruited and data on 416 are presented. Follow up is complete to 1/1/08 with 244 relapses after a median follow-up of 40 months. At complete remission (CR) following induction, a greater log reduction in WT1 transcripts was associated with a significantly reduced relapse risk even after adjustment for age, WBC, cytogenetics, performance status and de Novo/Secondary AML (hazard ratio (HR) 0.57 (0.46–0.71) per log reduction p<0.0001). The 5yr relapse rates (RR) for patients with a log reduction of <2, 2–3, and 3 or more were 77%, 50% and 26% respectively (p <0.00001). Furthermore, presence or absence of a 2 log reduction adds to the MRC risk index (developed to identify patients who might benefit from transplant or be candidates for experimental therapies, Burnett et al. ASH 2006, Abstract 18) for survival from CR (HR 0.59 (0.37–0.91), p=0.02). With respect to post induction WT1 transcripts, there were naturally three groups for both BM and PB levels for relapse risk: <1000, 1000–4999, 5000+ copies for BM (5 yr RR 45%, 74%, 82%, p<0.0001 ) and <1000, 1000–2999 and 3000+ copies for PB (5yr RR 53%, 82%, 91%, p<0.0001). After consolidation/SCT, BM and PB transcript levels were also highly predictive of relapse: BM <5000 copies RR 37%, BM 5000+ copies RR 84% (p<0.0001); PB <1000 copies RR 32%, PB 1000+ copies RR 90% (p<0.0001). Looking at all data together, in a multivariate analysis adjusted for MRC risk group, the most important prognostic variables for relapse, relapse free survival and overall survival were post induction log reduction and post chemotherapy PB transcript level >500 copies. In all cases PB transcript levels were more prognostic than levels from BM. We conclude that MRD monitoring in WT1 positive AML patients allows risk stratification based on treatment response, and in this group of patients, MRD monitoring adds to already known risk factors. Whether patients identified as being at high risk of relapse benefit from further treatment, including allogeneic stem cell transplantation, remains to be determined in future studies.

Detection and Treatment of Molecular Relapse in Acute Myeloid Leukemia with RUNX1, CBFB or MLL Gene Translocations. Frequent Quantitative Monitoring of Molecular Markers in Different Compartments and Correlation with WT1 Gene Expression.

Background. It seems that the population of leukemia stem cells (LSCs) have fundamental importance in the origin and maintenance of the acute myeloid leukaemia (AML). Eradication of LSCs is a new goal of AML therapy. We hypothesized, that by monitoring of minimal residual disease (MRD) and its dynamics in different compartments (peripheral blood, PB; bone marrow, BM; sorted CD34+ BM cells; and CD34- BM cells) it would be possible to find some patterns reliably predicting clinical relapse. Aims. To find which compartment is the best for MRD monitoring and whether it would be possible to treat the disease in the phase of molecular relapse in order to prevent the hematological relapse.Methods. MRD monitoring, in average once or twice per month, was performed in all phases of treatment, and was done even more frequently in the cases of unstable MRD. RQ PCR for fusion transcripts (CBFB/MYH11, RUNX1(AML1)/ETO or fusion transcripts of MLL gene) and WT1 gene was used. Molecular relapse was defined as reappearance of the fusion transcript detection or its 10-fold increase, repeatedly detected. Some patients with already known MRD dynamics and high probability of imminent hematological relapse were treated at the time of molecular relapse. Results. In 67 AML patients and 6 healthy volunteers, 2352 BM or PB samples were examined, including 265 samples from CD34+ BM cells. Follow up was 31–287 weeks (median: 113 w). The correlation between the fusion transcripts levels in BM and PB was excellent (r=0,9676). The correlation between WT1 PB and BM levels was far less satisfactory. Since the WT1values did not mostly reach zero values even if the level of fusion transcript was 0, we wanted to find some “normal” value for WT1. Using the ROC curves, however, we were not able to find any WT1 level being a confidential marker of molecular remission in either compartment (PB, BM, CD34− or CD34+). In relapsed patients, the time from molecular to haematological relapse was 8 – 79 days (median: 25 d). In the cases of subsequent development of haematological relapses, the levels of fusion transcript in CD34+ BM cells were one order of magnitude higher than in the BM or PB, even in the case of CD34- blasts. Nine patients were treated for 17 molecular relapses with following results: chemotherapy, CR=2, PR=3, NR=1; gemtuzumab ozogamicin, CR=3, PR=1, NR=3; IL-2±DLI, CR=3, NR=1 (PR was defined as a decrease in fusion transcript level at least 10-fold, CR as a decrease to 0). Patients with CD33- blasts at diagnosis did not respond to gentuzumab ozogamicin. Non-responsiveness to one treatment option did not mean non-responsiveness to another treatment.Conclusion: Frequent quantitative monitoring (especially in CD34+ BM cells) of fusion transcripts (in contrast to WT1) is useful for reliable prediction of haematological relapse in AML patients. PB seems to be sufficient for frequent outpatient MRD monitoring. Efficient targeting of LSCs will be essential for AML cure, however, the best method is currently not known. Some now available procedures are sometimes surprisingly successful.

Individualities in a flock of free-roaming greylag geese: Behavioral and physiological consistency over time and across situations

The concept of personality implies individual differences in behavior and physiology that show some degree of repeatability/consistency over time and across contexts. Most studies of animal personality, particularly studies of individuals' variation in physiological mechanisms, have been conducted on selected individuals in controlled conditions. We attempted to detect consistent behaviors as well as physiological patterns in greylag ganders ( Anser anser) from a free-roaming flock living in semi-natural conditions. We tested 10 individuals repeatedly, in a handling trial, resembling tests for characterization of “temperaments” in captive animals. We recorded the behavior of the same 10 individuals during four situations in the socially intact flock: (1) a “low density feeding condition”, (2) a “high density feeding condition”, (3) a “low density post-feeding situation” and (4) while the geese rested. We collected fecal samples for determination of excreted immuno-reactive corticosterone (BM) and testosterone metabolites (TM) after handling trials, as well as the “low density feeding” and the “high density feeding” conditions. BM levels were very highly consistent over the repeats of handling trials, and the “low density feeding condition” and tended to be consistent over the first two repeats of the “high density feeding condition”. Also, BM responses tended to be consistent across contexts. Despite seasonal variation, there tended to be inter-test consistency of TM, which pointed to some individual differences in TM as well. Aggressiveness turned out to be a highly repeatable trait, which was consistent across social situations, and tended to correlate with an individual's resistance during handling trials. Also, “proximity to the female partner” and “sociability” – the average number of neighboring geese in a close distance while resting – were consistent. We conclude that aggressiveness, “affiliative tendencies” and levels of excreted corticosterone and testosterone metabolites may be crucial factors of personality in geese.

Detection of Molecular Relapse of AML by Very Frequent Quantitative Monitoring of Different Molecular Markers (Fusion Transcripts and WT1) in Different Compartments (Peripheral Blood-PB, Bone Marrow-BM, and Selected CD34+ Bone Marrow Cells) and Its Treatment with Chemotherapy or Gemtuzumab Ozogamicin.

Patients and Methods. In order to find some markers, which reliably enable to predict clinical relapse in AML patients, we primarily focused on patients with the known fusion transcript (CBFB-MYH, AML1-ETO, or involving the MLL gene) and correlated this values with the WT1 for estimation of the true value of WT1 monitoring of disease behavior in a given patients. The value of different compartments for minimal residual disease (MDR) monitoring (PB, BM or CD34+ BM cells) was also analyzed. The study was prospective; in the case of MDR dynamics, the patients were actively called for earlier visit. This strategy could estimate the real time interval from MRD level increase to hematological relapse detection. In the interventional part of the study, the patients with already known MRD dynamics were treated at the time of molecular relapse.Results and Discussion. In 67 AML patients and 3 healthy volunteers, 2184 BM or PB samples were examined, including 240 samples from CD34+ BM cells. Follow-up was 31–252 weeks (median: 88 w). The correlation between the fusion transcripts levels in BM and PB was excellent (r=0.9676). The correlation between WT1 PB and BM levels was far less satisfactory. Since the WT1 values were frequently >0 even if the level of fusion transcript =0, we wanted to find some “normal” value for WT1. Using the ROC curves, however, we were not able to find any WT1 level being a confidential marker of molecular remission in either compartment (PB, BM or CD34+). Molecular relapse was defined as a reappearance of the fusion transcript detection or its 10-fold increase, repeatedly detected. The time from molecular to hematological relapse was 8–79 days (median: 25 d). In the cases of subsequent development of hematological relapses, the levels of fusion transcript in CD34+ BM cells were one order of magnitude higher than in the BM or PB, even in the case of CD34− blasts. Eight patients were treated for 13 molecular relapses with following results: chemotherapy, CR=2, PR=2; gemtuzumab ozogamicin, CR=3, PR=1, NR=3; IL-2±DLI, CR=2 (PR was defined as a decrease in fusion transcript level at least 10-fold). Patients with CD33− blast at diagnosis did not respond to gemtuzumab ozogamicin. Non-responsiveness to one treatment option did not mean non-responsiveness to another treatment.Conclusion: Fusion transcript monitoring enables reliable detection of molecular relapse in AML and high values in CD34+ BM cells signalize imminent hematological relapse (even in the case of CD34− blasts). PB is a suitable compartment for frequent monitoring. However, in some cases, relapse are fulminate, hardly allowing any intervention. WT1 does not seem to be a reliable marker for exact molecular relapse detection. AML at the stage of molecular relapse behaves similarly to AML at the frank hematological relapse: there are CRs, PRs, or NRs when using chemotherapy or gemtuzumab ozogamicin. AML with CD33− blasts at diagnosis does not seem to respond to gemtuzumab ozogamicin at the stage of molecular relapse. Success of AML therapy in the future seems to be dependant on efficient targeting the leukemia stem cell.

Evidence for Ineffective Erythropoiesis in Patients with Severe Sickle Cell Anemia.

Peripheral destruction of sickled erythrocytes is recognized as the central pathologic mechanism of SCD. Less well established is the contribution of ineffective erythropoiesis to the pathophysiology of SCD. Patients with SCD frequently develop mixed hematopoietic chimerism after nonmyeloablative stem cell transplantation (NMA-SCT). This provides an opportunity to directly compare the differentiation and survival of normal donor and SCD erythropoiesis in vivo. To this end, total genomic DNA (gDNA) and erythroid-lineage specific chimerism in peripheral blood (PB) and marrow (BM) were analyzed in 5 patients (3 with SCD, 2 with non-SCD) following NMA-SCT with HLA-matched donors. RBC chimerism was measured by beta-globin RNA pyrosequencing, which directly and quantitatively sequences coding region polymorphisms in beta-globin RNA that distinguish recipient and donor-derived erythroid precursors. The clinical characteristics of the 5 patients are shown in the table below. All patients developed mixed gDNA chimerism, ranging from 18–50%, between days 60–180, with no differences between PB and BM levels. BM erythroid precursor chimerism, determined by Y chromosome FISH and ABO staining in 4 of 5 patients, were identical to total BM gDNA chimerism. PB from all 3 SCD patients had a ~2 fold greater level of donor-derived beta-globin RNA compared with total gDNA. These levels were similar for the 2 non-SCD patients. The presence of ineffective SS erythropoiesis was directly revealed by analysis of BM, which similarly revealed higher expression of donor-derived beta-globin RNA relative to the degree of donor erythroid progenitor engraftment in Pts 1–3. These findings were not present in Pts 4–5. Chimerism was determined in purified marrow populations of immature (pro-and basophilic –– glycophorin [GYPA+, CD71+ hi]) versus mature (polychromatophilic, orthochromatophilic [GYPA+, CD71+dim] ) erythroblasts to determine the stage of maturation at which SS erythroblasts are lost. While non-SCD patients demonstrated similar rates of donor and host erythropoiesis, all 3 SCD patients demonstrated progressive intramedullary loss of SS erythroblasts with maturation, with enrichment of donor erythroid precursor chimerism appearing from the earliest stages of hemoglobinization. The presence of ineffective erythropoiesis in SCD explains the maturation advantage of AA or SA donor erythroid precursor cells over SS cells that allows for greater donor contribution to overall erythropoiesis following SCT. This is the first definitive in vivo demonstration of ineffective erythropoiesis in SCD, and supports the notion that NMA-SCT can be curative for SCD in the setting of stable donor engraftment. Further studies examining the underlying mechanism of ineffective erythropoiesis in SCD are under investigation.Patient clinical characteristicsPt 1Pt 2Pt 3Pt 4Pt 5Pt age/sex34/F52/M5/M45/M45/FPt dxSCDSCD+ myelomaSCDCLLHDDnr sickle genotype/sexSA/MSA/MSA/M––/F––/Mstem cell sourcePBSCPBSCBMPBSCCB% donor at d60–180PB gDNA2545505410BM gDNA2652504920Cells: erythroid progenitors2349––5022PB beta-globin RNA661001005018BM beta-globin RNA551001005415

Conditioning Regimens in Stem Cell Transplantations Facilitate Homing by Increasing Bone Marrow SDF-1 through Induction of SDF-1 Gene Transcription.

Stromal Derived Factor-1 (SDF-1) is a key regulator of engraftment. The clinical relevance of SDF-1 is suggested by the relation between in vitro migratory capacity of CD34-positive cells towards SDF-1 and engraftment after stem cell transplantation (SCT). For better insight in engraftment after SCT clarifying the determinants of SDF-1 is therefore important. We investigated SDF-1 responses after chemotherapy and irradiation of the murine stromal M210B4 cell line and in allogeneic SCT patients. SDF-1 protein levels were measured in conditioned medium (CM) of the M210B4 cell line, in peripheral blood plasma and bone marrow plasma by ELISA. The migration supportive capacity of CM was tested by allowing CD34-positive cells to migrate over fibronectin coated transwells for 4 hours. Furthermore, sdf-1 gene expression was investigated by means of a quantitative PCR using mRNA from M210B4 cells or from buffy coats of peripheral blood and bone marrow cells.Both irradiation and cytarabine treatment of M210B4 cells resulted in a significant ~2-fold increase in SDF-1 levels and a significant ~1.5-fold increase in migration of CD34-positive cells. Changes in protein levels correlated with those of mRNA: both treatments resulted in a significant ~2-fold increase in SDF mRNA copies.In agreement with in vitro results, a significant ~3-fold increase in SDF-1 protein levels was induced by in vivo myeloablative and non-myeloablative conditioning regimens. The increase in SDF-1 was higher in BM plasma as compared to PB plasma, thereby significantly increasing BM-PB gradients (BM minus PB SDF-1 levels: 11.0±3.5 after versus 4.0±2.8 ng before conditioning). Corresponding with protein levels, sdf-1 mRNA increased in bone marrow cells of 4/5 patients (~40-fold increase, from 0.3±0.3% to 9.7±15.2% relative to GAPDH expression, n=4 of whom 2 received myeloablative and 2 non-myeloablative regimens). Wide ranges of increase in SDF-1 message (ranging from 0.26 to 104-fold increase) and protein levels (1.26 to 5.16-fold increase) were observed, which may be accounted for by differences in conditioning regimens.We conclude that generation of an SDF-1 gradient over the blood-bone marrow barrier by conditioning regimens plays a role in engraftment. The introduction of reduced intensity stem cell transplantations challenged the idea that sublethal conditioning is a prerequisite for engraftment by preparing vacant niches. Our observation that both non-myeloablative and myeloablative regimens result in an increase in SDF-1 gradient, can be proposed as an explanation. Interestingly, the variation in increase of SDF-1 after different conditioning regimens indicate that homing efficiency may be improved by reconsidering the optimal moment of stem cell infusion and the agents used in conditioning regimens.

Urinary 1-hydroxypyrene as an indicator for assessing the exposures of booth attendants of a highway toll station to polycyclic aromatic hydrocarbons.

In this study, 32 booth attendants (the exposure group) and 21 in pre-job training to become booth attendants (the reference group) were randomly selected from a highway toll station. Personal PAH samplings were conducted on the exposure group on each day during the studied workweek. Pre-shift urinary 1-hydroxylpyrene levels (1-OHP) were measured on the first day of the workweek (BM(pre)) for both the exposure and reference groups, but the post-shift 1-OHP levels were measured on the last day of the workweek (BM(pest)) only for the exposure group. For the exposure group, we found that their mean total PAH exposure level (C(total PAHs)) was 11 400 ng/m3 and that their mean BM(post) was significantly higher than their mean BM(pre) (= 3.02 and 0.910 micromol of 1-OHP/mol of creatinine, respectively). In addition, the mean BM(pre) for the exposure group were higher than that for the reference group (= 0.410 micromol of 1-OHP/mol of creatinine). The above results suggest that vehicle exhaust significantly affects the booth attendants' 1-OHP levels. None of the three personal factors (age, work experience, and smoking habit), except for C(total PAHs), had a significant effect on predicting booth attendants' BM(inc) levels (= BM(post) - BM(pre)) (R2 = 0.57). The above results suggest that urinary 1-OHP could be a suitable biomarker for characterizing workers' PAH exposures. Similarly, we found that none of the three personal characteristics, except for the involved vehicle flow rates and vehicle types, had a significant effect on predicting booth attendants' BM(inc), levels (R2 = 0.60). The above result suggests that the traffic densities of various traffic types could be a suitable surrogate indicator for assessing workers' PAH exposures. Considering that the type of traffic designed for a given type of tollbooth is quite similar all over the world, the results obtained from this study, at least, could be served as a stepping-stone for providing a cheaper and convenient way for assessing traffic PAH exposures in the future.

Long-Term Follow-Up of Minimal Residual Disease in Leukemia Patients by Monitoring WT1 (Wilms Tumor Gene) Expression Levels

Thirty-one patients (27 with acute myeloid leukemia [AML], 2 with acute lymphocytic leukemia [ALL], and 2 with acute mixed lineage leukemia [AMLL]) treated with conventional chemotherapy (CHT) and 23 patients (13 AML, 5 ALL, and 5 with chronic myeloid leukemia [CML]) treated with allogeneic bone marrow transplantation (BMT) were monitored for WT1 expression levels in BM and peripheral blood (PB) by reverse transcriptase-polymerase chain reaction over a long-term period (mean, 29 months for CHT and 24 months for BMT). Sixteen of the patients in the CHT group and 3 in the BMT group who had achieved complete remission suffered clinical relapse. In 10 of these patients, WT1 expression that had returned to normal BM levels (< 10(-3); the WT1 expression level of K562 cells was defined as 1.0) after complete remission (CR) either gradually or rapidly increased again to abnormal levels 1 to 18 months (mean, 7 months) before clinical relapse became apparent. In another 9 patients, WT1 expression never returned to normal BM levels even after CR and the subsequent relapse was accompanied by a rapid increase in WT1 expression to levels higher than 10(-2) (10(-3) levels in PB). On the other hand, the remaining 35 patients (15 CHT and 20 BMT) maintained their CR. In 29 of these patients (11 CHT and 18 BMT), WT1 expression either gradually or rapidly decreased to normal BM levels, whereas in the other 6 (4 CHT and 2 BMT), low or very low levels of WT1 mRNAs (10(-3) to 10(-2) in BM and 10(-5) to 10(-3) in PB) remain detectable, but without any clinical signs of relapse. A clear correlation was found to exist between the minimal residual disease (MRD) detected in the paired BM and PB samples for all types of leukemias (AML, ALL, and CML), with MRD in PB being approximately one-tenth of that in BM. WT1 quantitation of 168 paired BM and PB samples showed that PB samples were superior to BM samples for the detection of MRD. We conclude that monitoring of WT1 expression levels in BM and PB makes it possible to rapidly assess the effectiveness of individual treatment and diagnose clinical relapse in the early stage for all leukemia patients regardless of the presence or absence of tumor-specific DNA markers.